Roche DNA连接酶

For life science research only. Not for use in diagnostic procedures.

FOR IN VITRO USE ONLY.

T4 DNA Ligase

From Escherichia coli NM 989 acc. to Murray

Poly (deoxyribonucleotide): poly (deoxyribonucleotide) ligase (AMP-forming), EC 6.5.1.1.

Cat. No. 481 220 100 units

Cat. No. 716 359500 units Store at Ϫ15 toϪ25° C Cat. No. 799 009500 units, high concentration

Product description

Volume activities Cat. Nos. 481 220, 716 359: ca. 1 × 103 units/ml;

Cat. No. 799 009: Ն 5 × 103 units/ml.

For lot-specific values see data label.

One unit of T4 DNA ligase is the amount of enzyme

activity that converts 1 nmol [32P] from pyrophosphate

into Norit-absorbable material in 20 min at 37° C (1).

One unit corresponds to 0.2 units determined in the

exonuclease III resistance assay (2).

Storage buffer20 mM Tris-HCl, 60 mM KCl, 1 mM EDTA, 5 mM

dithioerythritol, 50% glycerol (v/v), pH 7.5 (4° C).

Dilution buffer for the activity determination 50 mM Tris-HCl, 10 mM dithioerythritol, bovine serum albumin 500 ␮g/ml, pH 7.6 (25° C).

Specific activity approx. 3 × 103 units/mg.

For lot-specific values see data label.

PurityՆ3 ␮g of T4 DNA ligase migrate as a single band in

SDS- polyacrylamide gel electrophoresis according to

Laemmli (3).

Stability The undiluted enzyme solution is stable when stored

at Ϫ15 to Ϫ25° C.

Supplied buffer Ligation buffer for T4 DNA ligase, 10-times

concentrated: 660 mM Tris-HCl, 50 mM MgCl2,

10 mM dithioerythritol, 10 mM ATP, pH 7.5 (20° C). Properties and typical results:

T4 DNA ligase catalyzes the formation of phosphodi-

ester bonds between neighbouring 3´-hydroxyl- and

5´-phosphate ends in double-stranded DNA. Single-

stranded nicks in double-stranded DNA are also closed

by T4 DNA ligase.

In order to achieve optimal results, please consider the

following:

•The DNA to be ligated should be purified by phenol

extraction and ethanol precipitation. Resuspension

of DNA should be done in water or in a buffer

without EDTA.

•For insertion of DNA into plasmid vectors the vector

DNA should be dephosphorylated with alkaline

phosphatase.

•T4 DNA ligase can be completely inactivated by a

10 min incubation at 65° C.

•Heat inactivation should be done if the ligation

reaction mixture is used in experiments other than

transformation assays. Otherwise a drastic decrease

of transformed colonies is possible (factor >20).

•The molar of ratio of vector DNA to insert DNA

should be 1:3.

Activity in ligation of DNA-fragments

Assay principle: DNA-fragments with blunt or

overlapping ends are incubated with T4 DNA ligase in

1× ligation buffer and the mixture of the resulting

products is separated by agarose gel electrophoresis.

The extent of ligation can be determined by estimation

of the relative intensity of fluorescence in the gel bands

of substrate and product.

Ligation

of sticky ends

1 unit T4 DNA ligase joins more than 95% of 1 ␮g

Hind III digested ␭DNA in 30 ␮l 1 × ligation buffer after

incubation at 4° C for 16 h.

Ligation

of blunt ends

1 unit T4 DNA ligase joins more than 80% of 1␮g Rsa I

digested ␭DNA in 30 ␮l 1× ligation buffer after

incubation at room temperature for 16 h. >95% ligation

is observed in the presence of 15% PEG (w/v).

0999.D 22.10.859 1504R MB GPS